Double-stranded (ds) regions in RNA molecules provide targets for cleavage by cellular nucleases involved in the maturation and decay of cellular and viral RNAs. The long-range goal of this project is to determine the mechanism of enzymatic cleavage of dsRNA and its role in regulating cellular and viral RNA function and metabolism. We have developed an RNase III model wherein the dsRNA-binding motif stimulates cleavage through enhanced binding, and perhaps also through formation of the active enzyme dimer. RNase III activity is regulated by phosphorylation on serine, catalyzed by the bacteriophage T7 protein kinase. The T7 protein kinase also undergoes autophosphorylation with concomitant down-regulation of activity. RNase III is phosphorylated on at least two serines by the T7 protein kinase, which causes a 4-fold increase in catalytic activity. Knowing the sites of phosphorylation, in conjunction with enzymatic assays for dsRNA binding and cleavage, will provide new information on how phosphorylation regulates an RNA processing enzyme. The autophosphorylation of T7 on one or more serine residues is also of interest. The identified phosphoserines can then be changed to alanine, creating an autophosphorylation-resistant protein kinase which may exhibit sustained phosphorylation activity. Future biochemical studies will determine the other cellular targets of phosphorylation by the T7 protein kinase.